Myosin II plays fundamental roles in cytokinesis, cell migration, and cell shape changes during development. In all these settings, it is well established that dynamic localized assembly of myosin into the cytoskeleton is critical for its cellular contractile roles. Despite the importance of spatially and temporally regulated assembly, the signaling mechanisms that control localized assembly and disassembly of myosin II are not understood in any system. We are using the simple amoeba Dictyostelium discoideum as a model system for identifying signaling pathways that regulate myosin assembly. This simple amoeba displays forms of cellular motility, chemotaxis, and second messenger signaling similar to those displayed by motile mammalian cells such as neutrophils or macrophages. Myosin II assembly in this system is regulated by phosphorylation/dephosphorylation of a set of mapped threonine residues that lie near the tip of the myosin tail.Under previous funding, we focused on the biochemistry and cell biology of the enzyme myosin heavy chain kinase A (MHCK A), which participates in the in vivo control of myosin assembly via phosphorylation of the mapped target sites in the myosin tail. MHCK A is now recognized as the prototype for a highly novel family of protein kinases present in Dictyostelium and throughout the animal kingdom. We have now identified several additional Dictyostelium members of this kinase family; our preliminary data indicate that at least two of these are also MHC kinases. We have evidence for dynamic localization control of two of these kinases during chemotaxis, and we have evidence that lipid signaling pathways and acidic phospholipids may regulate the activity of these enzymes. Studies proposed for the next funding period will focus on the cellular roles of these enzymes, using gene targeting to understand the relative cellular roles of each kinase, and using domain dissection approaches to understand the mechanisms involved in the dynamic recruitment of these enzymes to the cell cortex and to address the mechanism of activation of these enzymes by acidic phospholipids. Genetic approaches will also be used to identify new genes that participate in the control of myosin II assembly and localization.